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The breast and ovarian cancer susceptibility gene BRCA1 encodes a tumor suppressor. BRCA1 protein, which is involved in DNA damage response, has been thought to be found primarily in cell nuclei. In the present investigation, immunohistological studies of BRCA1 protein in frozen breast cancer tissue and MCF7 and HeLa cell lines revealed BRCA1 expression in both nucleoli and nucleoplasmic foci. Immunoelectron microscopic studies of estrogen-stimulated MCF7 cells demonstrated BRCA1 protein localization in the granular components of the nucleolus. Moreover, immunofluorescence of BRCA1 and nucleolin double-labeling showed colocalization in both nucleoli and nucleoplasmic foci in breast tumor cells and asynchronously growing MCF7 and HeLa cells. Multiparameter analysis of BRCA1 and nucleolin in relation to cell cycle position (DNA content) showed expression during G1-S and persistence of BRCA1 during G2/M. After gamma-irradiation of MCF7 cells, BRCA1 protein dispersed from nucleoli and nucleoplasmic foci to other nucleoplasmic sites, which did not colocalize with nucleolin. Small interfering RNA-mediated knockdown of BRCA1 protein resulted in decreased immunofluorescence staining, which was confirmed by Western blotting. The observed colocalization of BRCA1 and nucleolin raises new possibilities for the nucleoplasm-nucleolus pathways of these proteins and their functional significance.

Germline mutations that inactivate BRCA1 are responsible for breast and ovarian cancer susceptibility. One possible outcome of genetic testing for BRCA1 is the finding of a genetic variant of uncertain significance for which there is no information regarding its cancer association. This outcome leads to problems in risk assessment, counseling and preventive care. The purpose of the present study was to functionally evaluate seven unclassified variants of BRCA1 including a genomic deletion that leads to the in-frame loss of exons 16/17 (Delta exons 16/17) in the mRNA, an insertion that leads to a frameshift and an extended carboxy-terminus (5673insC), and five missense variants (K1487R, S1613C, M1652I, Q1826H and V1833M). We analyzed the variants using a functional assay based on the transcription activation property of BRCA1 combined with supervised learning computational models. Functional analysis indicated that variants S1613C, Q1826H, and M1652I are likely to be neutral, whereas variants V1833M, Delta exons 16/17, and 5673insC are likely to represent deleterious variants. In agreement with the functional analysis, the results of the computational analysis also indicated that the latter three variants are likely to be deleterious. Taken together, a combined approach of functional and bioinformatics analysis, plus structural modeling, can be utilized to obtain valuable information pertaining to the effect of a rare variant on the structure and function of BRCA1. Such information can, in turn, aid in the classification of BRCA1 variants for which there is a lack of genetic information needed to provide reliable risk assessment.

Recent evidence suggests that the breast and ovarian cancer susceptibility gene product BRCA1 is involved in at least two fundamental cellular processes: transcriptional regulation and DNA repair. However, the mechanism of action of BRCA1 in either of these processes is still unknown. Here, we report the characterization of a disease-predisposing allele of BRCA1, identified in a family with several cases of ovarian cancer, coding for a protein that displays temperature-sensitive activity in transcriptional activation. The mutant protein differs from the wild type protein at a single amino acid, R1699W that occurs in a region at the N-terminal BRCT domain that is highly conserved among BRCA1 homologs. When the C-terminus of the mutant protein (aa 1560-1863) was fused to a heterologous GAL4 DNA-binding domain and expressed in yeast or mammalian cells, it was able to activate transcription of a reporter gene to levels observed for wild type BRCA1 at the permissive temperature (30 degrees C) but exhibited significantly less transcription activity at the restrictive temperature (37 degrees C or 39 degrees C). Our results indicate that the transcriptional activity of the R1699W mutant can be modulated as a function of temperature and provide a novel experimental approach which can be utilized to dissect the molecular mechanism(s) of BRCA1 in processes related to transcription.

BRCA1 is a tumor suppressor gene and germ line mutations account for the majority of familial cases of breast and ovarian cancer. There is mounting evidence that BRCA1 functions in DNA repair and transcriptional regulation. A major hurdle to dissect the role of BRCA1 is the lack of molecular reagents to carry out biochemical and genetic experiments. Therefore, we used random mutagenesis of the C-terminus of BRCA1 (aa 1560-1863) to generate temperature-sensitive (TS) mutants in transcription activation. We obtained 11 TS mutants in transcription that localized primarily to the hydrophobic core of the BRCT-N domain of BRCA1. One of the mutants, H1686Q, also displayed temperature-dependent transcription activation in human cells. These conditional mutants represent valuable tools to assess the role of BRCA1 in transcription activation.